Chromatography ligand comprising domain c from staphylococcus aureus protein a for antibody isolation

ABSTRACT

The present invention relates to a chromatography ligand, which comprises Domain C from  Staphylococcus  protein A (SpA), or a functional fragment or variant thereof. The chromatography ligand presents an advantageous capability of withstanding harsh cleaning in place (CIF) conditions, and is capable of binding Fab fragments of antibodies. The ligand may be provided with a terminal coupling group, such as arginine or cysteine, to facilitate its coupling to an insoluble carrier such as beads or a membrane. The invention also relates process of using the ligand in isolation of antibodies, and to a purification protocol which may include washing steps and/or regeneration with alkali.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a filing under 35 U.S.C. § 371 and claims priorityto international patent application number PCT/SE2007/000862 filed Sep.27, 2007, published on Apr. 3, 2008, as WO 2008/039141, which claimspriority to patent application number 0602061-4 filed in Sweden on Sep.29, 2006.

FIELD OF THE INVENTION

The present invention relates to the field of chromatography, and morespecifically to a novel affinity ligand which is suitable for use inantibody isolation. Thus, the invention encompasses affinity ligands assuch, a chromatography matrix comprising ligands according to theinvention, and a process of antibody isolation, wherein the ligandaccording to the invention is used.

BACKGROUND OF THE INVENTION

The term chromatography embraces a family of closely related separationmethods based on the contacting of two mutually immiscible phases,wherein one phase is stationary and the other phase is mobile. One areawherein chromatography is of great interest is in the biotechnologicalfield, such as for large-scale economic production of drugs anddiagnostics. Generally, proteins are produced by cell culture, eitherintracellularly or by secretion into the surrounding medium. Since thecell lines used are living organisms, they must be fed with a complexgrowth medium containing sugars, amino acids, growth factors, etc.Separation of the desired protein from the mixture of compounds fed tothe cells and from other cellular components to a sufficient purity,e.g. for use as a human therapeutic, poses a formidable challenge.

In such separation, in a first step, cells and/or cell debris is usuallyremoved by filtration. Once a clarified solution containing the proteinof interest has been obtained, its separation from the other componentsof the solution is often performed using a combination of differentchromatography steps, often based on different separation principles.Thus, such steps separate proteins from mixtures on the basis of charge,degree of hydrophobicity, affinity properties, size etc. Severaldifferent chromatography matrices, such as matrices for ion exchange,hydrophobic interaction chromatography (HIC), reverse phasechromatography (RPC), affinity chromatography and immobilized metalaffinity chromatography (IMAC), are available for each of thesetechniques, allowing tailoring of the purification scheme to theparticular protein involved. An illustrative protein, which is ofsteadily growing interest in the medical field, is immunoglobulinproteins, also known as antibodies, such as immunoglobulin G (IgG).

As in all process technology, an important aim is to keep the productioncosts low. Consequently, improved chromatographic techniques arefrequently presented, and the matrices are when possible reused.However, since each use of a chromatography matrix will leave certaintraces of the operation just performed, many different cleaningprotocols are available for cleaning and/or restoring the matrix intoits original form. Commonly known materials that need to be removed aree.g. non-eluted proteins and protein aggregates as well as potentiallyhazardous materials, such as virus, endotoxins etc, which may originatefrom the cell culture. The most commonly used cleaning is a simple washwith buffer. For a more efficient cleaning of the matrix, treatmentswith acid and/or base are frequently used. In order to even moreefficiently restore the matrix, an alkaline protocol known as CleaningIn Place (CIP) is commonly used. The standard CIP involves treatment ofthe matrix with 1M NaOH, pH 14. Such harsh treatment will efficientlyremove undesired fouling of the above-discussed kind, but may inaddition impair some chromatography matrix materials. For example, manyaffinity matrices, wherein the ligands are proteins or protein-based,cannot withstand standard CIP, at least not while maintaining theiroriginal properties. It is known that structural modification, such asdeamidation and cleavage of the peptide backbone, of asparagine andglutamine residues in alkaline conditions is the main reason for loss ofactivity upon treatment of protein in alkaline solutions, and thatasparagine is the most sensitive of the two. It is also known that thedeamidation rate is highly specific and conformation dependent, and thatthe shortest deamidation half times in proteins have been associatedwith the sequences—asparagine-glycine- and -asparagine-serine. See e.g.Gülich, Linhult, Nygren, Uhlen and Hober (2000) Journal of Biotechnology80, 169-178. Stability towards alkaline conditions can be engineeredinto a protein ligand.

Despite the documented alkaline sensitivity, protein A is widely used asa ligand in affinity chromatography matrices due to its ability to bindIgG without significantly, affecting the affinity of immunoglobulin forantigen. As is well known, Protein A is a constituent of the cell wallof the bacterium Staphylococcus aureus. Such Staphylococcus protein,known as SpA, is composed of five domains, designated in order from theN-terminus as E, D, A, B, and C, which are able to bind antibodies atthe Fc region, and a C-terminal region (or “X” region) that does notbind any antibodies. Jansson et al (Jansson, Uhlen and Nygren (1998)FEMS Immunology and Medical Microbiology 20, 69-78:“All individualdomains of staphylococcal protein A show Fab binding”) have later shownthat all the individual SpA domains also bind certain antibodies at theFab region.

U.S. Pat. No. 5,151,350 (Repligen) relates to cloning and expression ofthe gene coding for a protein A and protein A-like material. The cloningof this gene with its nucleotide sequence characterization enabled in1982 for the first time to obtain quantities of a protein A-likematerial and nucleotide sequence for cloning in various host-vectorsystems.

Since the production of protein A in a recombinant system wasaccomplished, further genetic manipulations thereof have been suggested.For example, U.S. Pat. No. 5,260,373 (Repligen) describes geneticmanipulation of recombinant protein A in order to facilitate theattachment thereof to a support, and more specifically to the couplingthereof via arginine. Further, U.S. Pat. No. 6,399,750 (PharmaciaBiotech AB) describes another recombinant protein A ligand, which hasbeen coupled to a support via cysteine.

However, in order to maintain selectivity and binding capacity, ProteinA chromatography matrices of the above-discussed kind need to be cleanedunder milder conditions than conventional CIP. In this context, it isunderstood that the cleaning is closely related to the lifetime of thechromatography matrix. For example, a sensitive matrix may be cleanedwith standard CIP, if a reduced performance is acceptable. Thus, effortshave been made to provide chromatography matrices which present theoutstanding properties, such as selectivity, of protein A, but which aremore resistant to alkaline conditions used for CIP.

Thus, U.S. Pat. No. 6,831,161 (Uhlen et relates to methods of affinityseparation using immobilized proteinaceous affinity ligands, wherein oneor more asparagine (Asn) residues have been modified to increasealkaline stability. This patent also describes methods of making astabilized combinatorial protein by modification of Asn residues withina protein molecule to increase stability of the protein in alkalineconditions, and randomization of a protein molecule to modify itsbinding characteristics, and combinatorial proteins wherein in a stepseparate from the randomization step, the stability of the protein inalkaline conditions has been increased by modifying one or more of itsAsn residues.

Further, WO 03/080655 (Amersham Biosciences) relates to animmunoglobulin-binding protein, wherein at least one asparagine residuehas been mutated to an amino acid other than glutamine or aspartic acid.According to this patent application, such more specific mutationconfers an increased chemical stability at pH-values of up to about13-14 compared to the parental molecule. The mutated protein can forexample be derived from a protein capable of binding to other regions ofthe immunoglobulin molecule than the complementarily determining regions(CDR), such as protein A, and preferably from the B-domain ofStaphylococcal protein A. The invention also relates to a matrix foraffinity separation, which comprises the described mutatedimmunoglobulin-binding proteins as ligands.

Despite the above-described development t ore alkaline-stable proteinA-based chromatography ligands, there is still a need in this field ofimproved ligands and chromatography matrices for highly specificisolation of antibodies, and of alternative wild type ligandconstructions that allow easier manufacture.

One example of such an improved chromatography matrix is described in US2006/0134805 (Berg et al), which relates to a separation matrixcomprised of porous particles to which antibody-binding protein ligandshave been immobilised. More specifically, the disclosed chromatographymatrix has been optimised in terms of ligand density; gel phasedistribution coefficient (Kay); and particle size to provide a matrixespecially suitable for high capacity purification of antibodies. Theligands of the disclosed matrix may comprise antibody-binding proteinsuch as Protein A, Protein G and/or Protein L.

SUMMARY OF THE INVENTION

One aspect of the present invention is to provide a novel chromatographyligand, which is capable of withstanding repeated cleaning-in-placecycles. This may be achieved by an affinity ligand which is based ondomain C from SpA Domain C, as defined in the appended claims.

Another aspect of the present invention is to provide an economicalprocess of purifying immunoglobulins. This may be achieved by a processwhich uses an affinity chromatography ligand capable of withstandingrepeated cleaning-in-place cycles.

Further aspects and advantages of the invention will appear from thedetailed disclosure that follows.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows results of testing the alkaline-stability of the ligandaccording to the invention as compared to other protein-based ligands.The X axis shows the incubation time in hours; while the Y axis showsthe capacity that remains after X hours in 0.5M NaOH, as described inExample 1. Mores specifically, the Protein A-containing productMABSELECT™ (♦); the more recent Protein A product MABSELECT SURE™,marketed as more alkaline-stable (X); Domain C from SpA as defined bySEQ ID NO 1 (Δ); and finally a deleted embodiment of Domain C from SpAas defined by SEQ ID NO 2 (▪). As appears from FIG. 1, the Domain Cligand according to the invention shows an alkaline-stability wellcomparable to the alkaline-stable product MABSELECT SURE™.

FIG. 2 shows the results of testing the Fab-binding properties of theligand according to the invention, as compared to other protein-basedligands. As appears from this figure, a chromatography ligand comprisingDomain C from SpA (Cwt and Cdel) present a much higher levels ofFab-binding than the other tested ligands.

DEFINITIONS

The term Domain C or “functional fragments or variants thereof”encompasses fragments or variants of SpA Domain C, which have theproperty of binding to IgG at the Fe region. The terms “antibody” and“immunoglobulin” are used interchangeably herein, and are understood toinclude also fusion proteins comprising antibodies and fragments ofantibodies.

The term an “Fe-binding protein” means a protein capable of binding tothe crystallisable part (Fe) of an antibody and includes e.g. Protein Aand Protein G, or any fragment or fusion protein thereof that hasmaintained said binding property.

The term “Fab fragment” refers to the variable part of an antibody;hence a “Fab-binding ligand” is capable of binding to either fullantibodies via Fab-binding; or to antibody fragments which includes thevariable parts also known as Fab fragments.

The term “chromatography” is used herein for any kind of separationwhich utilises the principles of chromatography, and hence includesbatch as well as HPLC methods.

The term “affinity chromatography” is used herein for the specific modeof chromatography where the ligand interacts with target via biologicalaffinity in a “lock-key” fashion. Examples of useful interactions inaffinity chromatography are e.g. enzyme-substrate interaction,biotin-avidin interaction, antibody-antigen interaction etc.

The term “protein-based” ligands means herein ligands which comprise apeptide or protein; or a part of a peptide or a part of a protein.

The term “isolation” of an antibody is used herein as embracingpurification of a specific product antibody from a mixture comprisingother proteins, such as other antibodies, and other components; as wellas the separation of an antibody from a product liquid, i.e. to removean undesired antibody.

DETAILED DESCRIPTION OF THE INVENTION

Thus, the present invention relates to a novel chromatography ligand.The chromatography ligand according to the invention, which isprotein-based and of the kind known as affinity ligand, comprises all orparts of Domain C from Staphylococcus protein A (SpA). In a firstaspect, the present invention relates to a chromatography ligand, whichligand comprises one or more Domain C units from Staphylococcus proteinA (SpA), or a functional fragment or variant thereof in one embodiment,the present chromatography ligand is substantially alkaline-stable. Inthis context, the term “substantially alkaline-stable” is understood tomean that the ligand is capable of withstanding repeatedcleaning-in-place cycles using alkaline wash liquid without loosing itsbinding capacity. In a specific embodiment, the present invention is achromatography ligand, comprises Domain C from Staphylococcus protein A(SpA), but none of the other domains of SpA.

In an alternative aspect, the present invention relates to achromatography ligand, which ligand comprises one or more Domain C unitsfrom Staphylococcus protein A (SpA), or a functional fragment or variantthereof, which chromatography ligand is capable of binding to the Fabpart of antibodies, as discussed in more detail below.

As discussed above, Jansson et al have already shown that Domain C canact as a separate immunoglobulin adsorbent, not just as part of ProteinA. The present inventors have confirmed that the immunoglobulin bindingproperties of Domain C are fully satisfactory for the use thereof as achromatography ligand. As also discussed above, Gülich and others hadshown that asparagine and glutamine residues in alkaline conditions isthe main reason for loss of protein A activity upon treatment inalkaline solutions, and that asparagine is the most sensitive of thetwo. Consequently, the Domain C ligand, which contains as many as sixasparagine residues, was not be expected to present any substantialalkaline-stability as compared to protein A.

However, as shown in the experimental part below, and in FIG. 1, thepresent inventors have quite surprisingly shown that the SpA Domain Cpresents a much improved alkaline-stability compared to a commerciallyavailable Protein A product (MABSELECT™, GE Healthcare, Uppsala, Sweden)by incubation in alkaline conditions for durations as long as 20 hours.In fact, the Domain C ligand presents values of alkaline-stability whichare similar to those of the product marketed as alkaline-stable(MABSELECT SURE™, GE Healthcare, Uppsala, Sweden), wherein asparagineresidues have been mutated to other amino acids.

In addition to this, as discussed above, it has been shown that anespecially alkaline-sensitive deamidation rate is highly specific andconformation dependent, and that the shortest deamidation half timeshave been associated with the sequences—asparagine-glycine- and-asparagine-serine. Quite surprisingly, the Domain C ligand of theinvention presents the herein presented advantageous alkaline-stabilitydespite the presence of one asparagine-glycine linkage between residues28 and 29, using the conventional numbering of the residues of Domain C.

In one embodiment, the ligand according to the invention is able toresist at least 10 hours in 0.5 M NaOH, without deviating more thanabout 10%, and preferably no more than 5%, from its originalimmunoglobulin binding capacity, Thus, after 5 hours, it will notdeviate more than 10%, preferably 5% from its original binding capacity.In other words, one embodiment of the present invention is a ligand asdescribed above, which after 5 hours incubation in 0.5M NaOH hasretained at least 95% of its original binding capacity.

In an advantageous embodiment, the ligand according to the invention isable to resist at least 15 hours in 0.5 M NaOH without loosing more thanabout 20%, and preferably no more than 10%, of its originalimmunoglobulin binding capacity. In a more advantageous embodiment, theligand according to the invention is able to resist at least 20 hours in0.5 M NaOH without losing more than about 30%, and preferably no morethan 15%, of its original immunoglobulin binding capacity. In otherwords, one embodiment of the present invention is a is a ligand asdescribed above, which after 15 hours incubation in 0.5M NaOH hasretained at least 80%, advantageously at least 90% of its originalbinding capacity.

The skilled person in this field can easily test alkaline-stability byincubating a candidate ligand with sodium hydroxide e.g. as described inthe experimental pad, and subsequent testing of the binding capacity byroutine chromatography experiments.

As easily realised by the skilled person in this field, a chromatographyligand according to the invention may consist of the wild type SpADomain C amino acid sequence, as shown in SEQ ID NO 1, herein denoted.Cwt. In an alternative embodiment, the chromatography ligand accordingto the invention consists of a functional fragment of SpA Domain C, suchas the one shown in SEQ TD NO 2, which discloses a sequence hereindenoted. Cdel, wherein Asn-Lys-Phc-Asn in positions 3-6 have beendeleted as compared to the wild type SpA Domain C sequence. In yet analternative embodiment, a variant of SpA Domain C is prepared by addingone or more amino acids e.g. to either end of the wild type SpA Domain Camino acid sequence; or by mutation of the wild type SpA Domain C aminoacid sequence, provided that such mutation does not substantiallyinterfere with the herein described properties relating toimmunoglobulin-binding and alkaline-stability Thus, in a specificembodiment, the chromatography ligand according to the inventioncomprises SpA Domain C, as shown in SEQ ID NO 1, which in additioncomprises the mutation G29A. Alternatively, the chromatography ligandaccording to this embodiment comprises the deleted SpA Domain C, asshown in SEQ ID NO 2, which consequently comprises said mutation inposition 25 (i.e. G25A). As the skilled person will recognise, suchaddition, mutation or deletion of amino acids as compared to the wildtype sequence should preferably not substantially affect the foldingpattern of the SpA Domain C ligand.

Thus, in one embodiment, the amino acid sequence of the ligand accordingto the present invention is the sequence defined by SEQ ID NO 1. In aspecific embodiment, the ligand according to the invention comprises atleast 60%, advantageously at least 80%, more advantageously at least 90%and most advantageously at least 95%, such as about 98% of the aminoacids shown in SEQ ID NO 1. In a specific embodiment, the ligandaccording to the invention comprises at least 35, advantageously atleast 46, more advantageously at least 52 and most advantageously atleast 55, such as 57, of the amino acids shown in SEQ ID NO 1.

In an alternative embodiment, the amino acid sequence of the ligandaccording to the present invention is the sequence defined by SEQ ID NO2. In a specific embodiment, the ligand according to the inventioncomprises at least 40%, advantageously at least 77%, more advantageouslyat least % and most advantageously at least 94%, such as about 98% ofthe amino acids shown in SEQ ID NO 2. In a specific embodiment, theligand according to the invention comprises at least 31, advantageouslyat least 42, more advantageously at least 48 and most advantageously atleast 51, such as 53, of the amino acids shown in SEQ NO 2.

As discussed in the section Background above, methods are readilyavailable for coupling of protein ligands via certain amino acids,preferably amino acids that contain nitrogen and/or sulphur atoms, seee.g. U.S. Pat. No. 6,399,750 or 5,084,559. Thus, in one embodiment, theligand according to the invention further comprises a terminal couplinggroup, said group preferably comprising one or more nitrogen and/orsulphur atoms. In an advantageous embodiment, the terminal couplinggroup is comprised of arginine or cysteine. In one embodiment, thecoupling group is in the C terminal region.

Further, the present invention also relates to a multimericchromatography ligand (also denoted a “multimer”) comprised of at leasttwo Domain C units, or a functional fragments or variants thereof, asdefined above. In one embodiment, this multimer comprises no unitsoriginating from SpA. In a specific embodiment, the multimer comprisesno other protein-based units. In another embodiment, the multimercomprises no other unit capable of any substantial interaction with atarget such as an antibody or a Fab fragment, thus it comprises no otherligand unit. As the skilled person in this field will realise, making amultimer may require adding one or more peptides as linkers between theunits. Thus, a multimer limited to containing only Domain C unitsaccording to the invention may in addition comprise linkers allowingconstruction of a multimer wherein each Domain C unit is sufficientlyexposed to be able to participate in the binding of target.

In another embodiment, the multimer comprises one or more additionalunits, which are different from Domain C and preferably protein-basedand equally alkaline-stable as Domain C. Thus, in the multimer, theligand according to the invention may be repeated and/or combined withother units from other sources, such as other proteins. In oneembodiment, the multimer is comprised of 2-8 units, such as 4-6 units.In one embodiment, one or more linker sequences are inserted between themultimer units. Such linkers may e.g. be inserted to allow the actualligand units to maintain their folding pattern. Linkers in this contextare well known, and the skilled person can easily decide on suitableamino acids and chain lengths which do not interfere with the hereindiscussed properties of the ligand. In a specific embodiment, thechromatography ligand according to the invention comprises no other SpAdomains than Domain C.

In a second aspect, the present invention relates to a nucleic acidsequence encoding a chromatography ligand as described above. Thus, theinvention encompasses all forms of the present nucleic acid sequencesuch as the RNA and the DNA encoding the ligand. The invention embracesa vector, such as a plasmid, which in addition to the coding sequencecomprises the required signal sequences for expression of the ligandaccording the invention. In one embodiment, the vector comprises nucleicacid encoding a multimeric ligand according to the invention, whereinthe separate nucleic acids encoding each unit may have homologous orheterologous DNA sequences. This aspect also embraces an expressionsystem comprising a nucleic acid sequence encoding a ligand according tothe invention. The expression system may e.g. be a prokaryotic host cellsystem, e.g. E. coli which has been modified to express the presentligand. In an alternative embodiment, the expression system is aeukaryotic host cell system, such as a yeast.

As the skilled person in this field will appreciate, the ligandaccording to the invention may alternatively be produced by proteinsynthesis methods, wherein the ligand is obtained by an automatedprocess adding amino acids one at a time following a predeterminedsequence. In an advantageous embodiment, segments of amino acids aminoacid sequences are synthesized and linked to each other to prepare theligand according to the invention. Such synthesis and linking proceduresare well known to the skilled person in this field.

In a third aspect, the present invention relates to a chromatographymatrix comprised of ligands as described above coupled to an insolublecarrier. Such a carrier may be one or more particles, such as beads orregular shapes; membranes; filters; capillaries; monoliths; and anyother format commonly used in chromatography. Thus, its an advantageousembodiment of the matrix, the carrier is comprised of substantiallyspherical particles, also known as beads. Suitable particle sizes may bein the diameter range of 5-500 μm, such as 10-100 μm, e.g. 20-80 μm. Inan alternative embodiment, the carrier is a membrane. To obtain highadsorption capacities, the carrier is preferably porous, and ligands arethen coupled to the external surfaces as well as to the pore surfaces.Thus, in an advantageous embodiment of the matrix according to theinvention, the carrier is porous.

The carrier may be made from an organic or inorganic material. In oneembodiment, the carrier is prepared from a native polymer, such ascross-linked carbohydrate material, e.g. agarose, agar cellulose,dextran, chitosan, konjac, carrageenan, gellan, alginate etc. The nativepolymer carriers are easily prepared and optionally cross-linkedaccording to standard methods, such as inverse suspension gelation (SHjertén: Biochim Biophys Acta 79(2), 393-398 (1964). In an alternativeembodiment, the carrier s prepared from synthetic polymer or copolymer,such as cross-linked synthetic polymers, e.g. styrene or styrenederivatives, divinylbenzene, acrylamides, acrylate esters, methacrylateesters, vinyl esters, vinyl amides etc. Such synthetic polymer carriersare easily prepared and optionally cross-linked according to standardmethods, see e.g. “Styrene based polymer supports developed bysuspension polymerization” (R Arshady: Chimica e L'Industria 70(9),70-75 (1988)). Native or synthetic polymer carriers are also availablefrom commercial sources, such as GE Healthcare Bio-Sciences AB, Uppsala,Sweden, for example in the form of porous particles. In yet analternative embodiment, the carrier is prepared from an inorganicpolymer, such as silica. Inorganic porous and non-porous carriers arewell known in this field and easily prepared according to standardmethods.

In a fourth aspect, the present invention relates to a method ofpreparing a chromatography matrix, which method comprises providingligands as described above; and coupling of said ligands to a carrier.In an advantageous embodiment, the coupling is carried out via anitrogen or sulphur atom of the ligand. In brief, the ligands may becoupled to the carrier directly; or indirectly via a spacer element toprovide an appropriate distance between the carrier surface and theligand. Methods for immobilisation of protein ligands to porous ornon-porous surfaces are well known in this field; see e.g. theabove-discussed U.S. Pat. No. 6,399,750.

In a fifth aspect, the present invention relates to a process ofisolating one or more target compounds, which process comprisescontacting a liquid comprising said compound(s) with a chromatographymatrix; allowing said compound(s) to adsorb to ligands present on thematrix, wherein said ligands consists of one or more Staphylococcusprotein A (SpA) Domain C, and/or functional fragments or variantsthereof; and, optionally, eluting said compound(s) by the passing acrosssaid matrix of a liquid that releases compound(s) from ligands. Thus, inthis embodiment, the ligands comprise no other. SpA-derived domain thanDomain C, or a functional fragment or variant thereof. In an alternativeembodiment, said ligands are multimers comprising two or more SpA DomainC units, or functional fragments or variants thereof.

In an advantageous embodiment, the ligands are the ligands describedabove. The target compound(s) may be any organic compound, bio moleculeor other biological material, such as proteins, e.g. antibodies;peptides; cells, such as eukaryotic and prokaryotic cells; nucleicacids, such as DNA, e.g. plasmids, and RNA; virus; etc. In anadvantageous embodiment, the target compound(s) is one or moremonoclonal or polyclonal antibodies, such as IgA, IgD, IgE, IgG, andIgM. In one embodiment, the target compound is a fragment of anantibody, such as a Fab fragment. In yet another embodiment, the targetcompound is a fusion protein wherein at least one part is an antibody oran antibody fragment.

In one embodiment, the chromatography matrix is a disposable product,and elution will then not be required if the purpose of the process isto remove the target compound such as the antibody from a productliquid. This embodiment may e.g. be for the removal of an undesiredantibody from a liquid, such as a medical liquid or a liquid whereinmany antibodies are produced, such as milk from a recombinant animal.

In an alternative embodiment, when the adsorbed compound is the desiredproduct, the elution step is included in the process. To obtain the mostsuitable conditions for adsorption, a liquid sample is combined with asuitable buffer or other liquid such as water to provide the mobilephase. The present method is advantageously run under conditionsconventional for affinity chromatography, and especially for protein Achromatography, as is well known in this field.

In a sixth aspect, the present invention relates to the use Domain C ofSpA, or a functional fragment or variant thereof, as alkaline-stableimmunoglobulin adsorbent. In this context, “alkaline-stable” isunderstood to mean that the adsorbent alkaline-stability is not lowerthan about 10%, such as about 5%, below that of a commercial productsmarketed as being alkaline-stable, such as MABSELECT SURE™ (GEHealthcare Bio-Sciences AB, Uppsala, Sweden) during the first 5 hours ofincubation in 0.5M NaOH. In an advantageous embodiment, the adsorbent isa ligand as described above. As said MABSELECT SURE™ should present aminimal deterioration after such time and conditions, the antibodybinding capacity of the adsorbent should not be lower than about 10%,such as about 5%, below its original binding capacity after such timeand conditions. In this context, the term “original” refers to itscapacity before any alkaline regeneration, and the comparisons arecarried out as side-by-side experiments using a procedure of the hereindisclosed kind.

In one embodiment, the use according to the invention comprises aprocess as described above, wherein the antibodies are eluted from thematrix and which is carried out at least once, such as 2-300 times,optionally with washing steps between; alkaline regeneration of thematrix; and finally repeating said process of isolating antibodies,Washing may be carried out with a suitable buffer, such as the bufferused to equilibrate the column. In an advantageous embodiment, theregeneration is carried out by incubation with 0.5 M NaOH.

The present invention also embraces a method of purifying one or moretarget compounds, as discussed above, which method comprises one or morechromatography steps in addition to the purification using thechromatography matrix according to the invention. The method accordingto this aspect may e.g. comprise s first chromatography step using thepresent matrix; an intermediate chromatography step using either ionexchange or hydrophobic interaction chromatography (HIC); and finally apolishing step using ion exchange, HIC or reverse phase chromatography.In a specific embodiment, this process comprises a step preceding thechromatography matrix having Domain C ligands as described herein. Sucha preceding step may e.g. be a conventional filtration, sedimentation,flocculation or other step to remove cell debris and other undesiredcomponents.

In an alternative embodiment, the use according to the invention is ananalytical or diagnostic use, such as an immunoassay.

EXAMPLES

The present examples are provided as illustrative purposes only, andshould not be construed as limiting the present invention as defined inthe appended claims.

Example 1: Column Study of the Alkaline Stability of Four ProteinA-Derived Ligands

In this example, the alkaline stability of four chromatography matrices,two of which were comparative and two of which were according to theinvention, were tested through a series of chromatographic runs:

-   -   MABSELEC™ and MABSELECT SURE™ (both comparative products        comprising protein-based ligands, GE Healthcare Bio-Sciences,        Uppsala, Sweden), and -Cwt (wild type Domain C from SpA, as        defined in SEQ NO. 1), and Cdel (deleted wild type Domain C from        SpA, as defined in SEQ ID NO. 2).

The IgG-binding capacity was measured initially and after incubationsteps in 0.5 M NaOH. The incubation times varied from one to five hours,with an accumulated incubation time of 20 hours.

The ligands according to the invention were immobilized on agaroseparticles according to standard procedure and packed in columns (GEHealthcare). Two of the matrices, MABSELECT™ and MABSELECT SURE™, arecommercial products manufactured by GE Healthcare marketed for thepurification of monoclonal antibodies. The ligands of both products arebased on the IgG binding Staphylococcus aureus Protein A. The MABSELECT™ligand basically is recombinant Protein A, which consists of fivehomologous domains (E, D, A, B, C). By comparison, the MABSELECT SURE™ligand consists of four domains which originate from the domain Banalogue “Z”, which in turn has been stabilized against high by proteinengineering methods. As a result, MABSELECT SURE™ toleratescleaning-in-place (CIP) conditions of up to 0.5 M NaOH. Both theMABSELECT™ and MABSELECT SURE™ ligands are coupled to agarose particles.

The ligands Cwt and Cdel were constructed as tetramers of identicaldomains with a C-terminal cysteine residue for coupling to a matrixaccording to standard procedure.

Materials & Methods Target Compound

10×10 ml injection liquid, solution, GAMMANORM® 165 mg/ml (Octapharmano. 00 86 64), human normal immunoglobulin, for subcutane infusion orintramuscular injection, was used as the target compound in thechromatography experiments.

Chromatography Columns

Ligand coupling and column packing was carried out as outlined in Table1 below:

TABLE 1 Columns used in Experiment 1 Column Column Column volumeLigand/Matrix ID no. Batch Date (ml) MABSELECT  9 4 U669082 2006 2.08SURE ™ Mar. 10 Cwt 11 2 U1555055A 2006 2.02 Mar. 10 MABSELECT ™  1 7U1555045A 2006 2.12 Mar. 10 Cdel 13 2 U1555059A 2006 2.06 Mar. 03

“Column ID” refers to a unique number given to each column. Thesenumbers were included in the chromatography methods and can be found inthe logbook of the result files. For example, the first column in table1 was called “MABSELECT SURE™ U669082 Column 4 20060310 (9)”. “Columnno.” is the packing number, i.e. columns packed with the same batch ofmatrix received different Column nos. upon packing. The column volumewas estimated by measuring the bed height.

Buffers and Solutions

Buffer A: 50 mM Sodium phosphate, 0.15 NI NaCl, pH 7.2Buffer B: 50 mM Citric acid, 0.15 M NaCl, pH 2.5

Instruments and Laboratory Equipment

Chromatography system: ÄKTA EXPLORER™ 10 (GE HealthcareColumn hardware: TRICORN™ Wm 5/100 GL (GE Healthcare)Vacuum degasser: CT 2003-2, 2 channel degasser, ChromTech AB

Spectrophotometer: NANODROP™ ND-1000 Spectrophotometer, NanoDropTechnologies

Centrifuge: Beckman Coulter AVANTI® J-20 XPI with JLA 8.1000 rotorpH meter (Buffer A): Beckman Φ 360 pH/Temp/mV MeterpH meter (Buffer B): Laboratory pH Meter CG 842, SCHOTT

Helium: AGA Gas AB, 10 l H 20577708, Instrument

Filter for buffer and sample: 75 mm Bottle Top Filter 500 ml, 0.2 μmpore size, NalgeneFilter for 0.5 M NaOH: 75 mm Bottle Top Filter—500 ml, 0.45 μm poresize, Nalgene

Software

ÄKTA EXPLORER™ 10 was controlled by UNICORN™ 5.01 (GE Healthcare). Apartfrom controlling the system during the chromatography runs, UNICORN™ wasused for method programming and evaluation of the results.

Buffer Preparation

Buffer A: Sodium dihydrogen phosphate and NaCl were dissolved in water,A pH meter was calibrated using pH 4, pH 7 and pH 10 standard buffers.pH was monitored adding NaOH(aq) to the buffer until pH reached 7.2. Thebuffer was filtered and degassed with helium prior use.

Buffer B: Citric acid and NaCl were dissolved in water. A pH meter wascalibrated, using pH 7 and pH 2 standard buffers. pH was monitored whileadding NaOH(aq) to the buffer until pH reached 2.5. The buffer wasfiltered and degassed with helium prior use.

Preparation of 0.5 M NaOH

NaOH(s) was dissolved in water to 0.5 M. The solution was filtered anddegassed with helium prior use.

Sample Preparation Experiment 1

30 ml Gammanorm (165 mg/ml) was diluted to 1 mg/ml with 4950 ml BufferA. The sample was filtered through 0.2 urn into a sterile 5 litrebottle.

Three 280 nm absorbance measurements were performed on the sample usingNANODROP™ spectrophotometer: 1.2573 AU, 1.2432 AU and 1.2101 AU. Meanabsorbance: 1.2369 AU.

The absorbance at 280 nm was also measured on ÄKTA EXPLORER™ 10. Thesample was pumped with the system pump through the system in bypassmode. A 10 min UV cell was used and the flow rate was 0.83 ml/min. Theabsorbance at 280 nm was 1510 mAU. This value was used as a referencewhen making capacity calculations.

Method Description

Normally, a CIP cycle for MABSELECT SURE™ involves 10-15 minutes contacttime of the CIP solution ally 0.1-0.5 M NaOH). To reduce the amount ofCIP cycles in this study, longer contact times were used. The columnswere incubated for 1, 2 and 5 hour intervals, with a total contact timeof 20 hours. This corresponds to 80 to 120 cycles with 10-15 minutescontact time.

Prior to the CIP incubations two initial capacity measurements wereperformed per column. After the capacity measurements the columns wereincubated in 0.5 M NaOH. After each CIP incubation, one capacitymeasurement per column was carried out.

Schematically, the experiment was designed as follows:

-   -   Two initial capacity measurements per column.    -   CIP incubation, 1 hour.    -   One capacity measurement per column.    -   CIP incubation, 2 hour.    -   One capacity measurement per column.    -   CIP incubation, 2 hour.    -   One capacity measurement per column.    -   CIP incubation, 5 hour.    -   One capacity measurement per column.    -   CIP incubation, 5 hour.    -   One capacity measurement per column.    -   CIP incubation, 5 hour,    -   One capacity measurement per column.

System Setup:

The experiments were carried out in room temperature. However, thesample was kept on ice to avoid microbial growth. To avoid the formationof air bubbles when the cold sample was heated to room temperature, adegasser was connected between the sample and the pump. The ÄKTAEXPLORER™ 10 was equipped with a 10 mm UV cell for JV detection.

Both buffer and sample was pumped through the system pumps. Followinginlets were used:

-   -   Sample: B pump (inlet B1)    -   Buffer A: A pump (inlet A11)    -   Buffer B: A pump (inlet A12)    -   0.5 M NaOH: A pump (inlet A13)

Capacity Measurement, Detailed Description

Prior to a capacity measurement (consisting of one capacity measurementper column) sample was pumped in bypass mode, i.e. no column used. Thepurpose of this was to get “fresh” sample to each capacity measurementand to avoid loading the first volume of sample that remained in tubesand the pump in room temperature during the CIP incubations, onto thefirst column.

The capacity measurement method for each column consisted of followingparts:

-   -   Equilibration of the column with 5 column volumes (CV) Buffer A.    -   Sample loading. Dynamic binding capacity is determined by        loading a sample onto a column packed with the chromatography        medium of interest. When the medium becomes more and more        saturated with sample, the level of absorbance at 280 nm will        increase due to unbound sample passing through the column. In        this method, the sample was loaded onto the column until the        UV_(280nm) curve reached 15% of the 280 nm absorbance of the        sample.    -   Wash out unbound sample. The column was washed with Buffer A        until the UV_(280nm) curve dropped below 10% of the 280 nm        absorbance of the sample    -   Elution. Bound material was eluted with 10 CV of Buffer B.    -   Reequilibration with 5 CV Buffer A.

The flow rate of sample loading was 0.83 ml/min.

CIP Incubation

After each capacity measurement, except for the first of the two initialmeasurements, a CIP incubation was carried out. In the CIP incubationmethod, 3 CV of 0.5 M NaOH was pumped through each column at a flow rateof 0.83 ml/min. After this the system was set to pause. The length ofthe pause depended on the length of the CIP incubation time, i.e. 1h, 2hor 5h. However, the time required for the system to pump NaOH throughthe columns was subtracted from the pause time. After a CIP incubation 3CV of Buffer A was pumped through each column at a flow rate of 0.83ml/min to remove the NaOH. By this procedure, all columns were exposedthe same amount of time to NaOH. One more wash cycle with 3 CV Buffer Awas finally carried out.

Evaluation of Chromatographic Results

Capacity was determined by measuring the volume of sample applied onto acolumn until the absorbance at 280 nm reached 10% of the sampleabsorbance. The dead volumes, i.e. the column volume, mixer and tubingfrom the pump to the UV cell, were subtracted from this volume. Thedelay volume without column was determined to 1.02 ml. The capacityvalues were plotted against the accumulated CIP incubation times.Relative capacity values were achieved by dividing the capacity valuesafter the CIP cycles with the mean of the start capacity values. Therelative capacity values were used for easier comparisons between thedifferent matrices.

TABLE 2 Results Experiment 1— Capacity (mg Gammanorm/ml chromatographymatrix(gel)) MABSELECT SURE ™ Cwt MABSELECT ™ Cdel Start 28.38 27.4328.98 30.86 Capacity 1 Start 28.13 27.40 28.98 30.95 Capacity 2 Capacityafter 29.32 27.79 26.98 30.75 1 h Capacity after 28.42 27.26 23.08 29.883 h Capacity after 28.25 26.94 20.30 29.25 5 h Capacity after 27.8826.07 15.79 26.87 10 h Capacity after 27.01 24.65 12.52 23.70 15 hCapacity after 25.93 23.02 10.14 20.35 20 h

Experiment 2: Test of Fab-Binding

The Fab-binding ability of the different chromatography media wasevaluated, in a 96-well filter plate assay. Liquids and chromatographymedia were mixed on a plate vortex instrument for 1 minute. The bottomof the wells consisted of a filter which retained liquids and theparticles of the chromatography media. When subjected to centrifugation,the liquids passed through the filter and were collected in a separate96-well collection UV-plate attached to the bottom of the filter plate.The absorbance at 280 nm of the collected liquid was measured in a platereader and used for detection and estimation of Fab. The liquids fromdifferent steps, e g washing, elution, were collected in differentplates and measured separately, to be able to measure the amount of Fabin individual fractions.

10% slurry was prepared of each chromatography medium.The filter plates were loaded with 200 μl slurry/well, i.e. 20 μlmedium/well.Equilibration—5×200 μl wash in PBSSample incubation—100 μl of human polyclonal Fab/Kappa, IgG fragment(Bethyl) in PBS, 15 minutes

Wash—5×100 μl PBS

Elution—3×100 μl 0.1 M glycine, pH 3.0CIP—2×10 min with 0.5 M NaOHAnalyze plates with liquids UV @ 280 nm

The results of experiment 2 are presented in FIG. 2.

The above examples illustrate specific aspects of the present inventionand are not in-tended to limit the scope thereof in any respect andshould not be so construed. Those skilled in the art having the benefitof the teachings of the present invention as set forth above, can effectnumerous modifications thereto. These modifications are to be construedas being encompassed within the scope of the present invention as setforth in the appended claims.

What is claimed is:
 1. A separation matrix comprising a plurality ofchromatography ligands coupled to a solid support, wherein the ligandcomprises one or more mutated Domain C of Staphylococcus protein A (SpA)and having a sequence that is at least 80% identical to the amino acidsequence shown in SEQ ID NO:1 or at least 77% identical to the aminoacid sequence shown in SEQ ID NO:2.
 2. The separation matrix of claim 1,wherein the ligands after 5 hours incubation in 0.5 M NaOH has retainedat least 95% of its original binding capacity.
 3. The separation matrixof claim 1, wherein the ligand is capable of binding to the Fab part ofan antibody.
 4. The separation matrix of claim 1, wherein the ligandcomprises one or more mutated Domain C of Staphylococcus protein A (SpA)and having a sequence that is at least 95% identical to the amino acidsequence shown in SEQ ID NO:1.
 5. The separation matrix of claim 1,wherein the ligand comprises one or more mutated Domain C ofStaphylococcus protein A (SpA) and having a sequence that is at least94% identical to the amino acid sequence shown in SEQ ID NO:
 2. 6. Theseparation matrix of claim 1, wherein the ligand comprises one or moremutated Domain C of Staphylococcus protein A (SpA) and having a sequencethat is at least 98% identical to the amino acid sequence shown in SEQID NO:1 or SEQ ID NO:
 2. 7. The separation matrix of claim 1, whereinthe one or more mutated Domain C of Staphylococcus protein A (SpA)comprises a sequence selected from SEQ ID NO: 1, SEQ ID NO: 2, SEQ IDNO: 1 with mutation G29A, and SEQ ID NO: 2 with mutation G25A.
 8. Theseparation matrix of claim 1, wherein at least one asparagine in thesequence of the ligand is replaced with an amino acid other thanglutamine or aspartic acid.
 9. The separation matrix of claim 1, whereinthe ligand comprises a terminal coupling group comprising at least onenitrogen and/or sulphur atom(s).
 10. The separation matrix of claim 9,wherein the terminal group comprises arginine or cysteine.
 11. Theseparation matrix of claim 1, wherein the ligand is coupled to the solidsupport via thioether bonds.
 12. The separation matrix of claim 1,wherein the ligand is multimeric that contains at least two Domain Cmutants.
 13. The separation matrix of claim 12, wherein the multimericligand comprises one or more other alkaline-stable protein-based units.14. The separation matrix of claim 12, wherein the multimeric ligandcomprises 2-8 Domain C units, optionally coupled via linker segments.15. The separation matrix of claim 1, wherein the solid support is apolysaccharide.
 16. The separation matrix of claim 1, wherein the solidsupport is comprised of substantially spherical particles.
 17. Theseparation matrix of claim 1, wherein the solid support is porous.
 18. Aseparation matrix comprising a plurality of chromatography ligandscoupled to a solid support, wherein the ligand comprises one or moremutated Domain C of Staphylococcus protein A (SpA) and having a sequenceselected from SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 1 with mutationG29A, and SEQ ID NO: 2 with mutation G25A.
 19. The separation matrix ofclaim 18, wherein the ligand comprises a terminal coupling group and theligand is coupled to the solid support via thioether bonds.
 20. Theseparation matrix of claim 18, wherein the solid support is porousspherical and comprises polysaccharide.